Stem cells that transform to beating cardiomyocytes

ABSTRACT

Disclosed herein is a novel isolated population of stem cells, called spoc cells, that can be induced, either in vivo or in vitro, to differentiate into cardiomyocytes. Methods are disclosed herein to differentiate the spoc cells, and to utilize these spoc cells for screening agents that affect cardiomyocytes. Methods are also provided herein to utilize spoc cells in therapeutic applications for the treatment of myocardial defects, such as areas of ischemic or traumatic damage.

FIELD

[0001] This application relates to the field of stem cells, specificallyto methods of producing and differentiating muscle stem cells.

BACKGROUND

[0002] Many Americans die each year of congestive heart failure. Heartfailure may occur from a variety of causes, including cardiomyopathy,myocardial ischemia, congenital heart disease, and valvular heartdisease, resulting in cardiac cell death and myocardial dysfunction. Ascardiomyocytes are not replaced in adult myocardial tissue, physiologicdemands on the existing, healthy, cardiomyocytes leads to theirhypertrophy. Heart transplants have been the only recourse for patientsin end-stage heart disease, however the United Network of Organ Sharing(UNOS), has reported that although more than 40,000 patients werewaiting for heart transplants as of February 2000, only 2,345 peoplereceived a donated heart in 1998. Furthermore, heart transplants arecomplicated by the incompatibility between the transplanted donor tissueand the recipient's immune system, which requires life-longimmunosuppression. Yet another drawback of heart transplants is theirhigh cost.

[0003] An alternative approach to heart transplantation is to generatecardiomyocytes from stem cells in vitro that can be used in thetreatment of heart failure, and other cardiac diseases characterized bymyocardial cell death or dysfunction. This approach is based on theability of stem cells to both self-renew and differentiate into one ormore mature cell types, including cardiomyocytes. Stem cells may beobtained from an individual suffering from heart disease and then usedto generate cardiomyocytes in vitro in order to repair the damagedmyocardium. This approach avoids problems inherent with hearttransplantation, such as lack of a suitable heart for transplant orimmune rejection of a transplanted heart.

[0004] Embryonic stem (ES) cells, derived from the inner cell mass ofthe blastocyst, are the most primitive stem cell, as disclosed in WO01/11011 A2. These cells have unlimited self-renewal capability, andbecause they can differentiate into several cell lineages and repopulatetissues upon transplantation, they have multipotent differentiativepotential. However, protocols are not available for differentiatingembryonic stem cells into beating cardiomyocytes.

[0005] Lineage specific stem cells, identified in most organ tissues,have less self-renewal capability than ES cells and theirdifferentiative ability is limited to tissues of that lineage. Of thelineage specific stem cells, the hematopoietic stem cell (HSC), derivedfrom bone marrow, blood, cord blood, fetal liver and yolk sack, is thebest characterized. These cells are defined by the expression of cellsurface markers, such as c-kit (c-kit+), and can terminallydifferentiate into all the hematopoietic cell types. HSC have been shownto contribute to the formation of functional cardiac tissue in vivo(Jackson et al, J. Clin. Invest., 107:1395-1402, 2001). Mesenchymal stemcells (MSC) are pluripotent progenitor cells derived from tissues ofmesodermal origin (U.S. Pat. No. 5,486,359). These cells are most oftenobtained from bone marrow, although they can be obtained from othersources, such as blood or dermis. These cells have been shown todifferentiate to form muscle, bone, cartilage, fat, marrow stroma andtendon, but have not been shown to differentiate into cardiomyocytes. Inaddition, progenitor cells have been identified in skeletal muscle,termed satellite cells (Cornelison and Wold, Dev. Biol., 191:270-283,1997). These cells are characterized by the expression of the cellsurface marker c-met (c-met+) in both its quiescent and activatedstates. When activated these cells re-enter the cell cycle, expressmyogenic regulatory factors, and differentiate into myoblasts.

[0006] However, despite the existence of a variety of stem cells, thereis currently no pure population of stem cells that can be induced underdefined conditions to differentiate into spontaneously beatingcardiomyocytes in vitro. Thus, there remains a need in the art forisolated populations of stem cells which can be induced to differentiateinto cardiomyocytes.

SUMMARY

[0007] The methods and cells described herein are based on the abilityof certain stem cells to be differentiated in vitro to form a fullyfunctional cell of more than one given cell type.

[0008] Disclosed herein is a novel isolated population of stem cells,called spoc cells, that can be induced, either in vivo or in vitro, todifferentiate into cardiomyocytes. Methods are disclosed herein todifferentiate the spoc cells, and to utilize these spoc cells forscreening agents that affect cardiomyocytes. Methods are also providedherein to utilize spoc cells in therapeutic applications.

[0009] The foregoing and other objects, features, and advantages willbecome more apparent from the following detailed description of aseveral embodiments which proceeds with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 is a series of digital images of transmission electronmicrographs demonstrating the progression of differentiation of CS(cardiac precursor from spoc cells) cells over time when cultured indifferentiation medium. FIG. 1A is a digital image of CS cell at day 3with disordered myosin filaments. FIG. 1B is a digital image showingthat at day 7 myosin filaments of characteristic 1.6 μm-length (top box)radiate outward and the cells contain dense bodies (lower box). FIG. 1Cand FIG. 1F are digital images of a cell at day 14, showing a single,central nucleus shows a stretching out of the dense bodies into anorganizing sarcomere. FIG. 1D shows that day 3 CS cells are round cellswith copious mitochondria (box and detail). FIG. 1E shows elongated day7 cells contain dense bodies (arrowhead). FIG. 1G shows that by day 56,a well-defined sarcomere (FIG. 1G) is present, with identifiable A- andI-bands and Z-lines.

[0011]FIG. 2 demonstrates the existence of calcium transients, incardiomyocytes differentiated from CS cells. FIG. 2A shows a graphicalrepresentation of the calcium transient in a beating CS cell-derivedcardiomyocyte. Peak intensity and baseline are shown in FIG. 2B and FIG.2C, respectively.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

[0012] In order to facilitate review of the various embodimentsdisclosed herein, the following list of abbreviations and explanation ofterms is provided:

[0013] I. Abbreviations and Terms

[0014] A. Abbreviations

[0015] CS: Cardiac precursors from spoc cells

[0016] DNA: Deoxyribonucleic acid

[0017] EGF: Epidermal growth factor

[0018] EGFP: Enhanced green fluorescent protein

[0019] ES: Embryonic stem

[0020] FACS: Fluorescence activated cell sort

[0021] FBS: Fetal bovine serum

[0022] FGF: Fibroblast growth factor

[0023] HSC: Hematopoietic stem cell

[0024] MRNA: Messenger ribonucleic acid

[0025] PBS: Phosphate buffered saline

[0026] RNase: Ribonuclease

[0027] RT-PCR: Reverse transcriptase-polymerase chain reaction

[0028] SPOC: Skeletal-based precursors of cardiomyocytes

[0029] B. Terms

[0030] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VI, published by Oxford UniversityPress, 1997 (ISBN 0-19-857778-8); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8)

[0031] Adult: A fully developed and physically mature subject, havingattained full size and strength.

[0032] Animal: Living multi-cellular vertebrate organisms, a categorythat includes, for example, mammals and birds.

[0033] Cardiac: Pertaining to the heart.

[0034] Cardiac dysfunction: Any impairment in the heart's pumpingfunction. This includes, for example, impairments in contractility,impairments in ability to relax (sometimes referred to as diastolicdysfunction), abnormal or improper functioning of the heart's valves,diseases of the heart muscle (sometimes referred to as cardiomyopathy),diseases such as angina and myocardial ischemia and infarctioncharacterized by inadequate blood supply to the heart muscle,infiltrative diseases such as amyloidosis and hemochromatosis, global orregional hypertrophy (such as may occur in some kinds of cardiomyopathyor systemic hypertension), and abnormal communications between chambersof the heart (for example, atrial septal defect). For furtherdiscussion, see Braunwald, Heart Disease: a Textbook of CardiovascularMedicine, 5th edition 1997, W B Saunders Company, Philadelphia Pa.(hereinafter Braunwald).

[0035] Cardiac muscle: The heart is made of specialized muscle tissuewith some similarities to both smooth and skeletal muscle. It isinvoluntary and mononucleate as is smooth muscle. Cardiac muscle isstriated like skeletal muscle, which means that it has microscopicallyvisible myofilaments arranged in parallel with the sarcomere. Thesefilaments slide along each other during the process of contraction inthe same manner as occurs in skeletal muscle. However, cardiac musclecontains more mitochondria so the striations are not as organized asthey are in skeletal muscle. Cardiac muscle also differs from skeletalmuscle in that the fibers in cardiac muscle branch and usually have asingle centrally located nucleus. Another difference in cardiac muscleis the presence of intercalated discs which serve as specializedconnections between cardiac muscle cells. These tight connections allowfor almost completely free movement of ions so that action potentialscan freely pass from one cell to another. This arrangement makes cardiacmuscle tissue a functional syncytium. When one cell is excited theresultant action potential is spread to all of them. This is animportant feature in that it allows the atrial or ventricular muscle tocontract as a unit to forcefully pump blood. Cardiac muscle can generateits own excititory impulses from the sino-atrial node, which acts like abiological pacemaker. In this manner, the contracting signal for cardiacmuscles originates in the heart itself. However, the autonomic nervoussystem (for example through the vagus nerve) can exert control over howfast the signals form and propagate through the heart, which regulatesthe rate of myocardial contraction. A “cardiomyocyte” is a cell of thecardiac muscle.

[0036] Cardiac precursors from spoc cells (CS cells): When spoc cellsare isolated from skeletal muscle and are cultured under growthconditions designed to promote their growth, spoc cells undergo severalrounds of division. During this proliferative phase they become clustersof floating round cells with an increased diameter as compared to spoccells. These round cells, with an increased diameter, are referred to asCS cells. In one embodiment, a diameter of a CS cell is from about 10 toabout 14 μm. When placed in growth promoting conditions in vitro (suchas the examples described below) CS cells differentiate intospontaneously beating cardiomyocytes.

[0037] Cardiomyopathy: Any disease or dysfunction of the myocardium(heart muscle) in which the heart is abnormally enlarged, thickenedand/or stiffened. As a result, the heart muscle's ability to pump bloodis usually weakened. The disease or disorder can be, for example,inflammatory, metabolic, toxic, infiltrative, fibroplastic,hematological, genetic, or unknown in origin. There are two generaltypes of cardiomyopathies: ischemic (resulting from a lack of oxygen)and nonischemic. Ischemic cardiomyopathy is a chronic disorder caused bycoronary artery disease—a disease in which there is atheroscleroticnarrowing or occlusion of the coronary arteries on the surface of theheart. Coronary artery disease often leads to episodes of cardiacischemia, in which the heart muscle is not supplied with enoughoxygen-rich blood. Eventually, the heart muscle enlarges from theadditional work it must do in the absence of sufficient oxygen-richblood.

[0038] Nonischemic cardiomyopathy is generally classified into threegroups based primarily on clinical and pathological characteristics:

[0039] (1) dilated cardiomyopathy, a syndrome characterized by cardiacenlargement and impaired systolic function of one or both ventricles;

[0040] (2) hypertrophic cardiomyopathy, herein defined as (a) global orregional increase in thickness of either ventricular wall or theinterventricular septum, or (b) an increased susceptibility to global orregional increase in thickness of either ventricular wall or theinterventricular septum, such as can occur in genetic diseases,hypertension, or heart valve dysfunction; or

[0041] (3) restrictive and infiltrative cardiomyopathies, a group ofdiseases in which the predominant clinical feature is usually impairedability of the heart to relax (diastolic dysfunction), and is oftencharacterized by infiltration of the heart muscle with foreignsubstances such as amyloid fibers, iron, or glycolipids.

[0042] See Wynne and Braunwald, The Cardiomyopathies and Myocarditities,Chapter 41, supra.

[0043] Cell surface marker: A protein, glycoprotein, or other moleculeexpressed on the surface of a cell, which serves to help identify thecell. A cell surface marker can generally be detected by conventionalmethods. Specific, non-limiting examples of methods for detection of acell surface marker are immunohistochemistry, fluorescence activatedcell sorting (FACS), or an enzymatic analysis.

[0044] Congenital heart disease: A heart-related problem that is presentsince birth and often as the heart is forming even before birth.Congenital heart disease may affect the heart, the heart's valves, theveins leading to, or the arteries leading away, from the heart, or theconnections between these parts of the body.

[0045] Differentiation: The process whereby relatively unspecializedcells (e.g., stem cells) acquire specialized structural and/orfunctional features characteristic of mature cells. Similarly,“differentiate” refers to this process. Typically, duringdifferentiation, cellular structure alters and tissue-specific proteinsappear. The term “differentiated muscle cell” refers to cells expressinga protein characteristic of the specific muscle cell type. Adifferentiated muscle cell includes a skeletal muscle cell, a smoothmuscle cell, and a cardiac muscle cell.

[0046] Differentiation Medium A synthetic set of culture conditions withthe nutrients necessary to support the growth or survival of culturedcells, and which allows the differentiation of stem cells intodifferentiated cells.

[0047] DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichcomprises the genetic material of most living organisms (some viruseshave genes comprising ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which comprises oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

[0048] Epidermal growth factor (EGF): In particular examples, EGF is aglobular protein of 6.4 kDa consisting of 53 amino acids. It containsthree intramolecular disulfide bonds essential for biological activity.EGF proteins are evolutionarily closely conserved. Human EGF and murineEGF have 37 amino acids in common. Approximately 70 percent homology isfound between human EGF and EGF isolated from other species. MammalianEGF includes, but is not limited to, murine, avian, canine, bovine,porcine, equine, and human EGF. The amino acid sequences and methods formaking these EGF polypeptides are well known in the art.

[0049] The gene encoding the EGF precursor has a length of approximately110 kb, and contains 24 exons. Fifteen of these exons encode proteindomains that are homologous to domains found in other proteins. Thehuman EGF gene maps to chromosome 4q25-q27.

[0050] EGF is a strong mitogen for many cells of ectodermal, mesodermal,and endodermal origin. EGF controls and stimulates the proliferation ofepidermal and epithelial cells, including fibroblasts, kidney epithelialcells, human glial cells, ovary granulosa cells, and thyroid cells invitro. EGF also stimulates the proliferation of embryonic cells.However, the proliferation of some cell lines has been shown to beinhibited by EGF.

[0051] EGF is also known to act as a differentiation factor for somecell types. It strongly influences the synthesis and turn-over ofproteins of the extra-cellular matrix including fibronectin, collagen,laminin, and glycosaminoglycans, and has been shown to be a strongchemoattractant for fibroblasts and epithelial cells.

[0052] EGF can be assayed in a cell-based assay wherein theproliferation of a cell population is assessed. EGF can also be assayedby an immunoassay, such as an ELISA assay.

[0053] Fragments of EGF, smaller than the full-length sequence can alsobe employed in methods disclosed herein. Suitable biologically activevariants can also be utilized. One specific, non-limiting example of anEGF variant of use is an EGF sequence having one or more amino acidsubstitutions, insertions, or deletions, wherein a biological functionof EGF is retained. Another specific, non-limiting example of an EGFvariant is EGF as wherein glycosylation or phosphorylation is altered,or a foreign moiety is added, so long as a biological function of EGF isretained. Methods for making EGF fragments, analogues, and derivativesare available in the art. Examples of EGF variants are known in the art,for example U.S. Pat. No. 5,218,093 and WO 92/16626A1. Examples of EGFfrom many different species are disclosed in WO 92/16626A1, as areexamples of variants, and strategies for producing them.

[0054] As used herein, “EGF” refers to naturally occurring EGF, andvariants and fragments that perform the same function of EGF in theculture media disclosed herein.

[0055] Embryonic stem (ES) cells are totipotent cells isolated from theinner cell mass of the developing blastocyst and can generate all of thecells present in the body (bone, muscle, brain cells, etc.). “ES cells”can be derived from any organism, for example from mammals such ashumans.

[0056] Fibroblast growth factor (FGF): Any suitable fibroblast growthfactor, derived from any animal, and functional variants and fragmentsthereof. A variety of FGFs are known and include, but are not limitedto, FGF-1 (acidic fibroblast growth factor), FGF-2 (basic fibroblastgrowth factor, bFGF), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6,FGF-7, FGF-8, and FGF-9. FGF refers to a fibroblast growth factorprotein such as FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, or FGF-9, or abiologically active fragment or mutant thereof. The FGF can be from anyanimal species. In one embodiment the FGF is mammalian FGF including butnot limited to, rodent, avian, canine, bovine, porcine, equine, andhuman. The amino acid sequences and method for making many of the FGFsare well known in the art.

[0057] Fragments of FGF that are smaller than those described can alsobe employed.

[0058] Suitable biologically active variants can be FGF analogues orderivatives. An analogue of FGF is either FGF or an FGF fragment thatincludes a native FGF sequence and structure having one or more aminoacid substitutions, insertions, or deletions. Analogs having one or morepeptoid sequences (peptide mimic sequences) are also included (see e.g.International Publication No. WO 91/04282). By “derivative” is intendedany suitable modification of FGF, FGF fragments, or their respectiveanalogues, such as glycosylation, phosphorylation, or other addition offoreign moieties, so long as the FGF activity is retained. Methods formaking FGF fragments, analogues, and derivatives are available in theart.

[0059] Growth factor: A substance that promotes cell growth, survival,and/or differentiation. In general, growth factors stimulate cellproliferation or maturation when they bind to their receptor. In oneembodiment, growth factors are a complex family of polypeptide hormonesor biological factors that control growth, division, and maturation ofmuscle cells. In another embodiment a growth factor can be used topromote the proliferation of muscle stem cells and maintain the stemcells in an undifferentiated state. A growth factor can be a naturallyoccurring factor or a factor synthesized using molecular biologytechniques. Examples of growth factors include platelet-derived growthfactor, fibroblast growth factor, epidermal growth factor, insulin,somatomedin, stem cell factor, vascular endothelial growth factor,granulocyte colony stimulating factor, and transforming growthfactor-beta, amongst others. A muscle cell growth factor is a growthfactor that effects the development (maturation), differentiation,division, or proliferation of muscle cells.

[0060] Growth medium: A synthetic set of culture conditions with thenutrients necessary to support the growth or survival of microorganismsor culture cells.

[0061] Heart: The muscular organ of an animal that circulates blood. Thewalls of the heart are comprised of working muscle, or myocardium, andconnective tissue. Myocardium is comprised of myocardial cells, whichare also referred to herein as cardiac cells, cardiac myocytes,cardiomyocytes and/or cardiac fibers. Cardiomyocytes may be cells of theatrium or cells of the ventricle.

[0062] Heart failure: The inability of the heart to supply sufficientoxygenated blood to meet the metabolic needs of the tissues and cells ina subject. This can be accompanied by circulatory congestion, such ascongestion in the pulmonary or systemic veins. As used herein, the termheart failure encompasses heart failure from any cause, and is intendedherein to encompass terms such as “congestive heart failure,” “forwardheart failure,” “backward heart failure,” “high output heart failure,”“low output heart failure,” and the like. See Chapters 13-17 inBraunwald for a detailed discussion. Conditions that could lead to heartfailure include, but are not limited to, coronary artery disease,cardiomyopathy, or congenital heart disease.

[0063] Heterologous: A heterologous sequence is a sequence that is notnormally (i.e. in the wild-type sequence) found adjacent to a secondsequence. In one embodiment, the sequence is from a different geneticsource, such as a virus or organism, than the second sequence.

[0064] Isolated: An “isolated” biological component (such as a nucleicacid molecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

[0065] Mammal: This term includes both human and non-human mammals.Similarly, the term “subject” includes both human and veterinarysubjects.

[0066] Muscle cell: Includes skeletal, cardiac or smooth muscle tissuecells. This term is synonymous with myocyte, and encompasses those cellswhich differentiate to form more specialized muscle cells (e.g.myoblasts). “Cardiomyocyte” refers to a cardiac muscle cell.

[0067] Myocardial injury: Damage to the muscle or the “myocardium” inthe wall of the heart as a result of disease or trauma. Myocardialinjury can be attributed to many things such as, but not limited to,cardiomyopathy, myocardial infarction, or congenital heart disease.

[0068] Nucleotide: “Nucleotide” includes, but is not limited to, amonomer that includes a base linked to a sugar, such as a pyrimidine,purine or synthetic analogs thereof, or a base linked to an amino acid,as in a peptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

[0069] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

[0070] Pharmaceutically acceptable carriers: Remington's PharmaceuticalSciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15thEdition (1975), describes compositions and formulations suitable forpharmaceutical delivery of stem cells herein disclosed.

[0071] In general, the nature of the carrier will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

[0072] Pharmaceutical agent: Refers to a chemical compound orcomposition capable of inducing a desired therapeutic or prophylacticeffect when properly administered to a subject or a cell. “Incubating”includes exposing a target to an agent for a sufficient period of timefor the agent to interact with a cell. “Contacting” includes incubatingan agent in solid or in liquid form with a cell.

[0073] Polypeptide refers to a polymer in which the monomers are aminoacid residues which are joined together through amide bonds. When theamino acids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred. The terms“polypeptide” or “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those which are recombinantlyor synthetically produced.

[0074] The term “polypeptide fragment” refers to a portion of apolypeptide which exhibits at least one useful epitope. The term“functional fragments of a polypeptide” refers to all fragments of apolypeptide that retain an activity of the polypeptide. Biologicallyfunctional fragments, for example, can vary in size from a polypeptidefragment as small as an epitope capable of binding an antibody moleculeto a large polypeptide capable of participating in the characteristicinduction or programming of phenotypic changes within a cell. An“epitope” is a region of a polypeptide capable of binding animmunoglobulin generated in response to contact with an antigen. Thus,smaller peptides containing the biological activity of insulin, orconservative variants of the insulin, are thus included as being of use.

[0075] The term “soluble” refers to a form of a polypeptide that is notinserted into a cell membrane.

[0076] The term “substantially purified polypeptide” as used hereinrefers to a polypeptide which is substantially free of other proteins,lipids, carbohydrates or other materials with which it is naturallyassociated. In one embodiment, the polypeptide is at least 50%, forexample at least 80% free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. In anotherembodiment, the polypeptide is at least 90% free of other proteins,lipids, carbohydrates or other materials with which it is naturallyassociated. In yet another embodiment, the polypeptide is at least 95%free of other proteins, lipids, carbohydrates or other materials withwhich it is naturally associated.

[0077] Conservative substitutions replace one amino acid with anotheramino acid that is similar in size, hydrophobicity, etc. Examples ofconservative substitutions are shown below. Original ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; GluMet Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe ValIle; Leu

[0078] Variations in the cDNA sequence that result in amino acidchanges, whether conservative or not, are usually minimized in order topreserve the functional and immunologic identity of the encoded protein.The immunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody; a variant that is recognized bysuch an antibody is immunologically conserved. Any cDNA sequence variantwill preferably introduce no more than twenty, and preferably fewer thanten amino acid substitutions into the encoded polypeptide. Variant aminoacid sequences may, for example, be 80, 90 or even 95% or 98% identicalto the native amino acid sequence. Programs and algorithms fordetermining percentage identity can be found at the NCBI website.

[0079] Precursor Cell: A cell that can generate a fully differentiatedfunctional cell of at least one given cell type. Generally, precursorcells can divide. After division, a precursor cell can remain aprecursor cell, or may proceed to terminal differentiation. A “muscleprecursor cell” is a precursor cell that can generate a fullydifferentiated functional muscle cell, such as a cardiomyocyte or askeletal muscle cell. One specific, non-limiting example of a muscleprecursor cell is a “cardiac precursor cell,” which is a cell that givesrise to cardiac muscle cells.

[0080] Progenitor Cell: A cell that gives rise to progeny in a definedcell lineage. A “muscle progenitor cell” is a cell that gives rise tocells of the muscle lineage. One specific, non-limiting, example of askeletal muscle progenitor cell is a “satellite cell,” which gives riseto immature and mature skeletal muscle cells.

[0081] Recombinant: A recombinant nucleic acid is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

[0082] Similarly, a recombinant protein is one encoded for by arecombinant nucleic acid molecule.

[0083] Skeletal muscle: Skeletal muscle makes up most of the body'smuscle and does not contract without nervous stimulation. It is undervoluntary control and lacks anatomic cellular connections betweenfibers. The fibers (cells) are multinucleate and appear striated due tothe arrangement of actin and myosin protein filaments. Each fiber is asingle cell, long, cylindric and surrounded by a cell membrane. Themuscle fibers contain many myofibrils that are made of myofilaments.These myofilaments are made up of contractile proteins. The key proteinsin muscle contraction are myosin, actin, tropomyosin and troponin.

[0084] Skeletal-based precursor of cardiomyocytes (Spoc) cells: Stemcells derived from skeletal muscle, which do not express the cellsurface markers c-met, or c-kit, that can be differentiated intocardiomyocytes. In one embodiment spoc cells are muscle derivedprecursor cells that are about 4 μm in diameter when cultured in vitro.These cells remain in suspension and proliferate when cultured in thepresence of a growth factor. Specific, non-limiting examples of growthfactors of use in propagating spoc cell are FGF, EGF, or a combinationthereof.

[0085] In one embodiment, spoc cells differentiate into spontaneouslybeating cardiomyocytes in vitro. During a proliferative phase (e.g.about 7 days after being maintained in vitro in the presence of a growthfactor), spoc cells cluster and increase in size to about 10-14 μm indiameter. The cells in these clusters, referred to as CS cells, have theability to differentiate into mature cardiac muscle cells when culturedin the absence of growth factors. Methods for isolating anddifferentiating spoc cells are disclosed herein.

[0086] Spontaneous: arising from an internal cause, resulting frominternal or natural processes, with no apparent external influence. A“spontaneously beating cardiomyocyte” is a cell that begins to beat as aresult of internal signals.

[0087] Stem cell refers to a cell that can generate a fullydifferentiated functional cell of more than one given cell type. Therole of stem cells in vivo is to replace cells that are destroyed duringthe normal life of an animal. Generally, stem cells can divide withoutlimit. After division, the stem cell may remain as a stem cell, become aprecursor cell, or proceed to terminal differentiation. Althoughappearing morphologically unspecialized, the stem cell may be considereddifferentiated where the possibilities for further differentiation arelimited. A “muscle stem cell” is a stem cell derived from muscle or thatgives rise to muscle cells after differentiation. One specific,non-limiting example of a muscle stem cell is a cell that gives rise tocardiac muscle cells.

[0088] Subject refers to any mammal, such as humans, non-human primates,pigs, sheep, cows, rodents and the like which is to be the recipient ofthe particular treatment. In one embodiment, a subject is a humansubject or a murine subject.

[0089] Suspension: a dispersion of solid particles, such as a cell,throughout the body of a liquid, such as a culture medium or an isotonic(physiologically compatible) buffer.

[0090] Therapeutic agent: Used in a generic sense, it includes treatingagents, prophylactic agents, and replacement agents.

[0091] Therapeutically effective amount is the amount of agent that issufficient to prevent, treat, reduce and/or ameliorate the symptomsand/or underlying causes of any of a disorder or disease. In oneembodiment, a “therapeutically effective amount” is sufficient to reduceor eliminate a symptom of a cardiac disease. In another embodiment, atherapeutically effective amount is an amount sufficient to overcome thedisease itself.

[0092] A therapeutically effective amount of a cell can be administeredin a single dose, or in several doses, for example daily, during acourse of treatment. However, the effective amount of the cells will bedependent on the subject being treated, the severity and type of thecondition, and the manner of administration of the compound.“Administering” can be accomplished by introducing the therapeuticallyeffective amount locally or systemically into the subject. Systemicintroduction can be accomplished by using an intravenous, intramuscular,transcutaneous or subcutaneous means. Such means could includeintroducing the therapeutically effective amount via injection, or viacatheter.

[0093] The general term “administering a therapeutically effectiveamount to the subject” is understood to include all animals (e.g.humans, apes, dogs, cats, horses, and cows) that have or may developsome form of cardiac dysfunction.

[0094] Transfected: A transfected cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Asused herein, the term transduction encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransduction with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

[0095] Transplantation: The transfer of a tissue or an organ, or aportion thereof, from one body or part of the body to another body orpart of the body.

[0096] Vector: A nucleic acid molecule as introduced into a host cell,thereby producing a transformed host cell. Recombinant DNA vectors arevectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. Viral vectors arerecombinant DNA vectors having at least some nucleic acid sequencesderived from one or more viruses.

[0097] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art. Although methods and materials similar or equivalentto those described herein can be used, suitable methods and materialsare described below. In case of conflict, the present specification,including the explanation of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Spoc Cells

[0098] Stem cells derived from skeletal muscle (spoc cells) aredisclosed herein. Spoc cells do not express the cell surface markersc-met, or c-kit, and are thus termed a c-met−/c-kit− cell. Spoc cellscan be isolated from any age mammal, either human or non-human. Thusspoc cells can be obtained from a fetus, a child or an adult of anymammalian species. In one embodiment, a spoc cell is human or murinec-met−/c-kit− cell that can be differentiated into a cardiomyocyte invitro. In one embodiment, the spoc cell is between about 3 μm and 10 μmin diameter, or are about 4 μm in diameter.

[0099] Culture conditions for spoc cells have been identified and aredisclosed herein. In one embodiment, spoc cells do not adhere to theculture dish but remain in suspension when cultured in the presence ofat least one growth factor. In one specific, non-limiting example, thegrowth factor is EGF. In another specific, non-limiting example, thegrowth factor is FGF.

[0100] Culture conditions are also disclosed herein (see below) fordifferentiating spoc cells. The differentiation of spoc cells intocardiomyocytes can be assessed by observing morphological changes. Insome examples, differentiated spoc cells are spontaneously beatingcardiomyocytes. In several embodiments, organized gap junctions andsarcomeres with clear Z-lines and A- and I-bands, are observed in thedifferentiated spoc cells. In addition, certain examples of thedifferentiated spoc cells may be mono- or multi-nucleate. In oneembodiment the cells are bi-nucleate.

[0101] The isolated spoc cell can be transduced using standardprocedures known in molecular biology in order to introduce a nucleicacid molecule of interest into the cell. In one embodiment, the nucleicacid molecule encodes a polypeptide. The polypeptide encoded by thenucleic acid molecule can be from the same species as the cells(homologous), or can be from a different species (heterologous). Forexample, a nucleic acid molecule can be utilized that supplements orreplaces deficient production of a peptide by the tissue of the hostwherein such deficiency is a cause of the symptoms of a particulardisorder. In this case, the cells act as a source of the peptide. In onespecific, non-limiting example the polypeptide is the cardiac specifictranscription factor GATA-4.

[0102] In one embodiment, the nucleic acid molecule of interest encodesa polypeptide involved in growth regulation or neoplastic transformationof cardiac cells. Specific, non-limiting examples of nucleic acidssequences of interest are SV40 Tag, p53, myc, src, and bcl-2. In anotherembodiment, the nucleic acid sequence of interest encodes an enzyme.Specific, non-limiting examples of enzymes are proteins involved in theconversion of a pro-drug to a drug, or growth factors that promote theexpansion, differentiation, or survival of cardiac progenitor cells,such as EGF, FGF, or atrial natriuretic factor. In yet anotherembodiment, the nucleic acid sequence of interest encodes atranscriptional regulator.

[0103] In one embodiment, the nucleic acid sequence of interest isoperably linked to a regulatory element, such as a transcriptionaland/or translational regulatory element. Regulatory elements includeelements such as a promoter, an initiation codon, a stop codon, mRNAstability regulatory elements, and a polyadenylation signal. A promotercan be a constitutive promoter or an inducible promoter. Specificnon-limiting examples of promoters include the CMV promoter, an atrialnatriuretic factor promoter, and promoters including TET-responsiveelement for inducible expression of transgene. In another embodiment,the nucleic acid sequence of interest is inserted into a vector, such asan expression vector. Procedures for preparing expression vectors areknown to those of skill in the art and can be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989). Expression of the nucleic acidof interest occurs when the expression vector is introduced into anappropriate host cell.

[0104] In yet another specific, non-limiting example, a nucleic acidsequence can be introduced to decrease rejection. For example, theimmunogenicity of a cell may be suppressed by deleting genes thatproduce proteins that are recognized as “foreign” by the host (aknock-out), or by introducing genes which produce proteins, such asproteins that are native to the host and recognized as “self” proteinsby the host immune system.

[0105] Thus in one embodiment, a spoc cell may be transfected with anucleic acid molecule designed to functionally delete or “knock-out” agene of interest. In this method, the nucleic acid molecule of interestis a nucleic acid molecule that undergoes homologous recombination andis inserted into the genome of the spoc cell. Methods for producing“knock-outs” in ES cells are known to one of skill in the art (e.g. seeU.S. Pat. No. 5,939,598).

[0106] According to this example, cells are cultured in vitro asdescribed herein and an exogenous nucleic acid is introduced into thecells by any method known to one of skill in the art, for example, bytransfection or electroporation. The transfected cultured cells can thenbe studied in vitro or can be administered to a subject (see below).Methods for the introduction of nucleic acid sequences into stem cellsare known in the art (e.g., see U.S. Pat. No. 6,110,743).

Methods of Isolating and Expanding Muscle Stem Cells

[0107] A method of isolating a c-met−/c-kit− cardiomyocyte precursorcell of muscular origin (spoc cell) is described herein. In this method,spoc cells are separated by size from a suspension of muscle cells andthe cells are cultured on a solid substrate. The cells that remain insuspension in the culture medium are isolated.

[0108] The method of isolation of the spoc cells includes obtaining thecells from the muscle of a subject. Muscle tissue can be prepared forthe purpose of isolating or obtaining individual spoc cells by usingmethods well known to one of skill in the art. Examples of methods oftissue preparation include enzymatic digestion with enzymes such ascollagenase, mechanical disruption using instruments such as hand-heldor motor-driven homogenizers, or by chemical disruption using, forexample, chelators of calcium and magnesium.

[0109] The preparation of muscle cells can be sorted by any method thatseparates cells on the basis of cell size. In one embodiment, the spoccells are isolated by passing digested skeletal muscle through a seriesof filters of varying pore size. The cells are passed through twofilters, where a first filter has a pore size of about 50-200 μm, about60-150 μm, about 80-100 μm, or about 100 μm and a second filter has apore size of about 10-50 μm, 20-40 μm, or about 40 μm. In one embodimentthe isolated cells are less than 40 μm in diameter. In otherembodiments, isolated cells are between about 3 μm and 10 μm indiameter. In another embodiment the isolated cells are about 4 μm indiameter.

[0110] The cells can be also sorted by size by passing them throughsize-exclusion columns. In one such embodiment, the cells are elutedalong a size gradient such that the largest cells are eluted first andthe smallest cells are eluted last. The cells can also be sorted by sizeusing FACS. Cells of about 3 μm to 10 μm in diameter, or of about 4 μmin diameter, are isolated.

[0111] Once the muscle cells are sorted by size the cells are furtherselected and then expanded in culture medium. In one embodiment thecells are cultured on a solid substrate that permits the adhesion of asubpopulation of cells in the presence of a culture medium. In oneembodiment, the solid substrate is a container, such as a tissue culturedish. In another embodiment, the solid substrate is in the form of beadsdesigned for tissue culture. The medium can be a growth medium, or anybuffer that maintains the viability of the cells. A variety of culturemedia are known and are suitable for use. Generally, the growth mediumincludes a minimal essential medium. In one embodiment, the medium isDMEM and/or F12, or a combination of DMEM and F12 (at a ratio betweenabout 1:1 to about 10:1).

[0112] The growth medium may be supplemented with serum. Specific,non-limiting examples of serum are horse, calf or fetal bovine serum.The medium can have between about 3% by volume to about 10% by volumeserum, or about 5% by volume serum.

[0113] In one embodiment, the medium contains one or more additionaladditives such as nutrients. Specific, non-limiting examples of thesenutrients are shown in the table below: Additive Exemplary Concentrationserum About 3% to about 10% insulin About 5 μg/ml to about 10 μg/mltransferrin About 5 μg/ml to about 10 μg/ml selenium About 6 ng/mlethanolamine About 2 μg/ml EGF About 5 ng/ml to about 10 ng/ml FGF About5 ng/ml to about 10 ng/ml gentamycin About 25 μg/ml to about 50 μg/mlfungizone About 0.2 μg/ml to about 2.5 μg/ml

[0114] The muscle stem cell growth media can also be supplemented withgrowth factors. In one embodiment, the growth medium includes basicfibroblast growth factor (bFGF). In one specific example, the growthmedium includes between about 2 ng/ml to about 100 ng/ml of bFGF, suchas for example between about 5 ng/ml to about 50 ng/ml, between about 8ng/ml to about 20 ng/ml, or between about 5 to about 10 ng/ml bFGF. Inyet another example, the medium includes about 10 ng/ml bFGF. In anotherembodiment, the growth medium includes epidermal growth factor (EGF). Inone specific example, the growth medium includes between about 2 ng/mlto about 100 ng/ml of EGF, such as for example between about 5 ng/ml toabout 50 ng/ml, between about 8 ng/ml to about 20 ng/ml, or betweenabout 5 ng/ml to about 10 ng/ml EGF. In yet another example, the mediumincludes about 10 ng/ml EGF. Thus in one embodiment, the growth mediumis 1:1 DMEM/F 12 and includes 5% fetal bovine serum, 10 ng/ml FGF, 10ng/ml EGF, 5 μg/ml insulin, 5 μg/ml transferrin, 6 ng/ml selenium, 2μg/ml ethanolamine.

[0115] In one specific, non-limiting example the cells are cultured inthe growth medium for about 4 days to about 8 days. In another specific,non-limiting example, the cells are cultured in the growth medium forabout 6 days to about 7 days.

[0116] During the period that the cells are cultured in the presence ofgrowth factors, the cells cluster and increase in size. Within theclusters the cells are between about 5-20 μm in diameter, or betweenabout 10-14 μm in diameter.

[0117] A method is also provided for isolating spoc cells wherein thespoc cells are identified using specific binding agents, such asantibodies, for example monoclonal antibodies that recognize cellsurface markers. This particular method of isolation of the spoc cellsincludes obtaining the cells from the muscle of a subject, as describedabove. In one embodiment, the cells are selected by size (see above) andthen the c-met−/c-kit− spoc cells are identified using the specificbinding agents, such as antibodies that recognize the c-met and c-kitcell surface markers.

[0118] In one embodiment the c-met and c-kit antibodies are immobilized.A particular embodiment uses magnetic cell sorting. This method involvesa combination of monoclonal antibodies which are covalently bound to thesurface of magnetic beads and which are directed to cell surface markerswhich are absent from the cells being selected. For example, to isolatethe c-met−/c-kit− spoc cells, monoclonal antibodies to c-met and c-kitbound to magnetic beads are used. All cells expressing either c-met, orc-kit, or both c-met and c-kit, will be bound by the antibodies andretained by the beads. Since the cells bound to the magnetic beads areimmobilized by the magnet, the c-met−/c-kit− cells that remain insuspension can be isolated from the other cells.

[0119] In another embodiment, purified populations of c-met−/c-kit− spoccells are isolated via FACS. Fluorescent-tagged antibodies against c-metand c-kit identify c-met+, c-kit+ and c-met+/c-kit+ double-positivepopulations of cells, allowing for the identification and isolation ofthe double-negative c-met−/c-kit− population.

[0120] In other embodiments a single antibody, or a combination ofantibodies, can be covalently bound to inert beads, such as sepharosebeads. The beads can be packed in a column or maintained as a slurry.The cells expressing one or more of the cell surface markers arerecognized by one or more of the antibodies, thus becoming bound to thebeads, thereby identifying a subpopulation of unbound cells that doesnot express the combination of cell surface markers.

[0121] In another embodiment the antibodies are not immobilized. In aparticular embodiment the addition of the antibodies to a mixture ofcells causes the aggregation of cells expressing the cell surfacemarkers recognized by the antibodies. The cells not expressing the cellsurface markers are excluded from the aggregates and can be isolated.

[0122] Spoc cells isolated by these or other methods can be maintainedin culture. The spoc cells can further be differentiated intocardiomyocytes.

Methods of Differentiating Muscle Stem Cells

[0123] A method is disclosed herein for differentiating a spoc cell intoa cardiomyocyte. In a particular example, the cardiomyocyte is aspontaneously beating cardiomyocyte.

[0124] In one embodiment, differentiation into cardiomyocytes is inducedby culturing cells in medium similar to the growth medium, but whichdoes not include at least one growth factor. Thus, a specific,non-limiting example of a differentiation medium is a growth medium thatlacks at least one growth factor. Growth factors removed from the mediuminclude, but are not limited to, bFGF or EGF, or a combination of bFGFand EGF.

[0125] Removal of at least one growth factor causes the cells to adhereto the tissue culture dish and acquire characteristics of adifferentiated cardiomyocyte. Differentiation refers to the processwhereby relatively unspecialized cells, such as the c-met−/c-kit−muscle-derived stem cells acquire specialized structural and/orfunctional features characteristic of mature cells, such ascardiomyocytes.

[0126] Differentiation of c-met−/c-kit− muscle stem cells intocardiomyocytes, such as spontaneously beating cardiomyocytes, can bemeasured by any method known to one of skill in the art. Specific,non-limiting examples are immunohistochemical analysis to detectexpression of cardiac polypeptides (e.g. troponin-T, L-type calciumchannel, or cardiac-specific transcription factors GATA-4, or Nkx2.5),or assays such as ELISA assay and Western blot analysis. Differentiationof cells can also be measured by assaying the level of mRNA coding forcardiac polypeptides using techniques such as Northern blot, RNaseprotection and RT-PCR. In another embodiment, the number ofspontaneously beating cells is assessed.

[0127] Calcium transients, or the flux in intracellular calciumconcentrations, can be used as a measure of cardiomyocytedifferentiation. In one embodiment calcium imaging is used to measurecalcium transients. For example, ratiometric dyes, such as fura-2,fluo-3, or fluo-4 are used to measure intracelluar calciumconcentration. The relative calcium levels in a population of cellstreated with a ratiometric dye can be visualized using a fluorescentmicroscope or a confocal microscope. In other embodiments, the membranepotential across the cell membrane is monitored to assess calciumtransients. For example, a voltage clamp is used. In this method, anintracellular microelectrode is inserted into the cardiomyocyte.

[0128] In one embodiment, calcium transients can be seen beforeobservable contractions of the cardiomyocytes. In other embodimentscalcium transients are seen either during, or after, observablecontractions of cardiomyocytes. In another embodiment the cells arecultured in the presence of conditions wherein the cells do not beat,such as in the presence of a calcium chelator (e.g. EDTA or EGTA) andthe calcium transients are measured.

Methods for Treatment of Cardiac Diseases or Disorders

[0129] In other embodiments, methods are provided for treating a subjectsuffering from a disease or a disorder, such as myocardial injury, oralleviating the symptoms of such a disorder, by administering cellsisolated and cultured according to the methods disclosed.

[0130] In one embodiment, spoc cells are isolated as described hereinand a therapeutically effective amount of spoc cells is administered tothe subject. In another embodiment, spoc cells are isolated anddifferentiated into cardiomyocytes, as disclosed above, and atherapeutically effective amount of the differentiated cells areadministered to a subject, such as a human. The cells may beadministered in any fashion, for example in a dose of, for example0.25-1.0×10⁶ cells. Different dosages can of course be used depending onthe clinical circumstances. The cells may be administered systemically(for example intravenously) or locally (for example directly into amyocardial defect under echocardiogram guidance, or by directapplication under visualization during surgery). In another example, thecells are administered in a gel matrix (such as Gelfoam from UpjohnCompany) which polymerizes to form a substrate in which the administeredcells can grow.

[0131] In one embodiment the subject has a myocardial injury. Themyocardial injury may be due to trauma that occurred as the result of anobject or projectile, such as a knife or a bullet, having penetrated themyocardium, or as a consequence of surgery to remove, for example, atumor. Myocardial injury may also result from diseases such ascardiomyopathy, myocardial infarction, or congenital heart disease. Inanother embodiment the subject is suffering from cardiac dysfunctionwhich includes, for example, abnormal or improper functioning of theheart valves, or abnormal communication between the chambers of theheart.

[0132] In one embodiment the spoc cells or differentiated cardiomyocytesare administered systemically by injection. Specific, non-limitingexamples include administration by subcutaneous injection, intramuscularinjection, or intravenous injection. If administration is intravenous,an injectible liquid suspension of spoc cells can be prepared andadministered by a continuous drip or as a bolus.

[0133] In another embodiment, the spoc cells or differentiatedcardiomyocytes are administered locally. One specific, non-limitingexample of local administration is intra-cardiac muscle injection. Forintra-cardiac injection, the spoc cells are in an injectible liquidsuspension preparation or in a biocompatible medium which is injectiblein liquid form and becomes semi-solid at the site of damaged myocardium.A conventional intra-cardiac syringe or a controllable endoscopicdelivery device can be used so long as the needle lumen or bore is ofsufficient diameter (e.g. 30 gauge or larger) that shear forces will notdamage the spoc cells.

[0134] In other embodiments the cells are administered locally on asupport medium. One specific, non-limiting example of a support mediumis a sterile mesh, or matrix, upon which the cardiomyocytes arecultured. A layer of cardiomyocytes, for example a confluent layer ofcardiomyocytes, cultured on such a matrix can be applied locally, orgrafted at or near, a site of myocardial injury. In one embodiment thesupport medium is a biodegradable mesh. In another embodiment thesupport medium is not biodegradable. The size of the mesh, and thedensity of cells on it, can vary depending on the myocardial defectbeing treated.

[0135] In another embodiment the cells are encapsulated prior toadministration, such as by co-incubation with a biocompatible matrixknown in the art. A variety of encapsulation technologies have beendeveloped (e.g. Lacy et al., Science 254:1782-84, 1991; Sullivan et al.,Science 252:7180712, 1991; WO 91/10470; WO 91/10425; U.S. Pat. No.5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538). Duringopen surgical procedures, involving direct physical access to the heart,all of the described forms of spoc cell delivery preparations areavailable options.

[0136] The cells can be repeatedly administered at intervals until adesired therapeutic effect is achieved.

Use of Spoc Cells Produced to Screen Agents that Affect CardiomyocyteDifferentiation or Function

[0137] In other embodiments, methods are provided for screening agentsthat affect cardiomyocyte differentiation or function. According to thismethod, a population of cardiomyocytes or their precursors is producedas described above. The population of cells is contacted with an agentof interest, and the effect of the agent on the cell population is thenassayed. The effect on differentiation, survival, proliferation, orfunction of the cells is assessed.

[0138] The methods described herein can be used to assess the effect ofan agent on cardiomyocyte differentiation. In order to assess the effectof a test agent on cardiomyocyte differentiation or function, the agentis contacted either to spoc cells or CS cells. In several embodimentsthe spoc cells are maintained in medium including a growth factorbetween about 1 day to about 8 days, between about 4 days to about 7days, or about 7 days before the addition of an agent.

[0139] In another embodiment the growth factor is removed from themedium, generating CS cells, at or before the agent is added. In severalspecific, non-limiting examples CS cells are maintained in the mediumbetween about 1 day to about 56 days, between about 7 days to about 28days, or between about 14 days to about 21 days before the addition ofan agent.

[0140] Differentiation of spoc cells contacted with an agent can beassessed by any means known to one of skill in the art. In oneembodiment the morphology is examined, for example electron microscopyis used to assess the ultrastructure of the cells. Suitable parametersfor evaluation include, but are not limited to the evaluation of gapjunctions between contacting cardiomyocytes. In other embodiments,immunohistochemical or immunofluorescence techniques are used to assessdifferentiation. In yet another embodiment, differentiation is assessedby analysis expression of specific mRNA molecules expressed incardiomyocytes. Suitable assay systems include, but are not limited toRT-PCR, in situ hybridization, Northern analysis, or RNase protectionassays. In a further embodiment the levels of polypeptides expressed indifferentiated cardiomyocytes are assayed. Specific, non-limitingexamples of polypeptide assays of use include Western blot analysis,ELISA assay, or immunofluorescence. Alternatively, calcium transientsare measured, as described above.

[0141] The assay can also be used to screen the effect of an agent oncardiomyocyte function. Any method known to one of skill in the art canbe utilized to assess cardiac function. In one embodiment the beatingrate of a cardiomyocyte is assayed to identify agents that increase ordecrease beating. One method for assessing the beating rate is toobserve beating under a microscope. Agents that can be screened in thismanner include inotropic drugs, such as sympathomimetic agents.

[0142] In one embodiment, cells contacted with the agent are comparedwith a control. Suitable controls include spoc or CS cells not contactedwith the agent, or contacted with vehicle alone. Standard values canalso be used as a control.

Kits

[0143] The cells described herein are ideally suited for the preparationof a kit. The kit can include a carrier means, such as a box, a bag, orplastic carton. In one embodiment the carrier contains one or morecontainers such as vials, tubes, and the like that include a sample ofspoc cells. In another embodiment, the carrier includes a container withan agent that affects differentiation, a buffer, or a vehicle for theintroduction of the cells. Instructions can be provided to detail theuse of the components of the kit, such as written instructions, videopresentations, or instructions in a format that can be opened on acomputer (e.g. a diskette or CD-ROM disk). These instructions indicate,for example, how to administer the cells to treat a myocardial defect orhow to use the cells to screen test agents of interest (such asinotropic drugs).

[0144] Without further elaboration, it is believed that one skilled inthe art can, using this description, utilize the present invention toits fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES Example 1 Method of Isolating and Expanding CardiomyocytePrecursor Cells from Adult Mouse Skeletal Muscle

[0145] Skeletal muscle tissue from hind legs of 6-10 week-old maleC57B1/SJ6 mice was cut into small pieces and digested with collagenasefor two hours at 37° C. The digested tissue was cleared of cell debrisand other undigested tissue fragments by passage through a 100 μm filterand then through a 40 μm filter (Falcon). The cell suspension wascentrifuged at low speed (1,400 rpm) to clear as much as of the smallmuscle fiber fragments as possible. The cells at this stage consistedmostly of clusters of small round cells approximately 4 μm in diameter,called spoc (skeletal-based precursors of cardiomyocytes) cells.

[0146] The spoc cells were plated at a density of approximately 10⁵cells per cm² in regular tissue culture dishes in complete growth medium(1:1 DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 ng/mlhuman EGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 μg/ml insulin, 5μg/ml transferrin, 6 ng/ml selenium, 2 μg/ml ethanolamine (ITS-X,Invitrogen Corporation), 25 μg/ml gentamicin and 2.5 μg/ml fungizone(Life Technologies)). After a few days, the culture consisted of afloating population of round cells and some adherent fibroblasts. Theround cells enlarged as they underwent a few rounds of cell divisionduring which time they became clusters of floating round cells with anincreased diameter of 10-14 μm. The cells in these clusters, werereferred to as CS (cardiac precursors from spoc) cells.

Example 2 Method of Differentiating Spoc Cells into Cardiomyocytes

[0147] CS cells were gently collected after seven days of growth incomplete growth medium. The cells were then plated in the same medium inthe absence of EGF and bFGF (differentiation medium) and were maintainedat 37° C. To assess the progression of differentiation of the cells, thecultures were observed at various time points using an inverted lightmicroscope. Beating frequency measurements of the cardiomyocytes wereobtained by video microscopy.

[0148] Under the differentiation culture conditions the cells graduallybegan to attach to the culture dish, and elongate in shape, taking onthe appearance of myoblasts. Within a few days of being maintained inthe differentiation medium, the cells began spontaneously beating.Elongated uninucleate cells (60 μm in length) and round uninucleatecells (15 μm in diameter) both exhibited spontaneous beating. By fourdays post replating the beating cells were more numerous. The beatingcells did not undergo any more cell divisions and were maintained inthis medium for several weeks, with the maintenance of the spontaneousbeating phenotype. Spontaneous beating was continuous and measured at afrequency of 1-8 Hz. Small contractions observed in a day 14 cell (30 μmin length) were likely the consequence of an immature contractileapparatus (FIG. 1C). Cells kept at room temperature beat continuouslyfor at least 3 hours.

Example 3 Detection of Cardiac-Specific Polypeptides byImmunofluorescence

[0149] The specimens were air-dried for 30 minutes and then fixed in 4%paraformaldehyde at 4° C. followed by a rinse for 5 minutes withphosphate buffered saline (PBS). They were blocked with goat serum for30 minutes and then incubated overnight, at 4° C., with either GATA-4(mAb H-112, Santa Cruz Biotechnology), sarcomeric myosin (MF-20 Ab,ATTC), cardiac-specific troponin-T (mAb RDI-TRK4T19-1A11, MolecularProbes, Inc.), cardiac L-type calcium channel (mAB AB5412-2000U1a,Chemicon Inc.), cardiac-specific transcription factor Nkx2.5 (mAb N-19,Santa Cruz Biotechnology), or connexin 43 (mAb 71-07000, ZymedLaboratories Inc.) (1:200). Following the overnight incubation, thespecimens were rinsed 3 times (5 minutes each) with PBS and blockedagain with goat serum for 30 minutes. The specimens were then incubatedat room temperature with a secondary antibody, conjugated with eitherFluorescein Isothiocyanate (FITC), Texas Red, or TetramethylrhodamineIsothiocyanate (TRITC), for 1 hour. They were again rinsed 3 times (5minutes each) with PBS and then visualized with a laser confocalmicroscope (Leica) to detect fluorescent signals.

[0150] The earliest time of GATA-4 expression is after 3 days in culturein growth factor containing medium. Within 3 days after replating thecells in differentiation medium, some cells begin to express sarcomericmyosin. Cytospins of day 7 CS cells stained with monoclonal antibodiesdemonstrate the expression of cardiac-specific transcription factorGATA-4, sarcomeric myosin, and cardiac-specific troponin-T. Day 14 cellsstained for GATA-4 and sarcomeric myosin. Overlays of images of cellsstained with GATA-4 and sarcomeric myosin demonstrated that they wereco-localized in the cell. At this early stage in development some cellsmay either be positive for GATA-4 or sarcomeric myosin. By day 28, themajority of cells express both proteins. By day 21 the cells arepositive for cardiac L-type calcium channel, cardiac-specifictranscription factor Nkx2.5, and connexin 43.

Example 4 Ultrastructure of Differentiated Cardiomyocytes

[0151] For routine transmission electron microscopy, cells were fixed insitu on Petri dishes with 1.25% glutaraldehyde in 0.1 M cacodylatebuffer containing 1% CaCl₂ at 4° C. for 2 hours. Following fixation,cells were washed three times in Sabatini's solution (0.1 M cacodylatebuffer containing 6.8% sucrose), and post-fixed with 1% osmium tetroxidein cacodylate buffer for one hour. After three washes in Sabatini'ssolution, samples were dehydrated in alcohol and embedded in Scipoxy 812(Energy Beam Sciences, Inc. Agawarm, Mass.). Polymerization was carriedout at 37° C. for 24 hours and then at 60° C. overnight. Ultra-thinsections were cut with a Leica Ultracut UCT ultramicrotome, stained withuranyl acetate and Reynold's lead citrate, and examined with a JEOL 1200CXII transmission electron microscope.

[0152] In FIG. 1, transmission electron micrographs show the progressionof CS cells. At day 3 round cells with disordered myosin filaments (FIG.1A) and large central nuclei surrounded by copious mitochondria (FIG.1D, box and detail) exist. By day 7 elongated cells (FIG. 1E) containdense bodies (FIG. 1E arrowhead and FIG. 1B, lower box). Myosinfilaments of characteristic 1.6 μm-length (FIG. 1B, top box) radiateoutward. A day 14 cell (FIGS. 1C and F) with a single, central nucleusshows a stretching out of the dense bodies (FIG. 1C) into an organizingsarcomere. By day 56, a well-defined sarcomere (FIG. 1G) is present,with identifiable A- and I-bands and Z-lines.

Example 5 Calcium Transients as a Measure of CardiomyocyteDifferentiation

[0153] Cardiomyocytes were incubated for 30 minutes at 37° C. withfluo-3 or fluo-4 dye at a concentration of approximately 5-10 μm inDMEM/F-12 (dyes dissolved in DMSO 1:1 with pluronic solution). The cellswere then washed with fresh DMEM/F-12. The images were collected with aZeiss LSM-510 laser scanning confocal system and a C-Apochromat63×objective (1.2 N.A.). Fluo-3 and fluo-4 were excited at 488 nm withan argon laser and the emission light was collected using an LP 505filter. The pinhole was adjusted to produce a 5 μm slice to minimize theinfluence of axial movements with contraction on viewing the calciumtransients. All transmitted light images were collected simultaneouslyusing a transmitted light detector in conjunction with the 488 nmexcitation light. Data depth for the images was 8-bit.

[0154] Calcium transients can be observed with confocal microscopy influo-3- and fluo-4-treated cells (FIG. 2). Fluorescent intensity isproportional to the amount of calcium binding to fluo-3 dye upon releaseof calcium from the sarcoplasmic reticulum. FIG. 2A shows a graphicalrepresentation of the calcium transient in a beating CS cell-derivedcardiomyocyte. Peak intensity and baseline are shown in FIG. 2B and FIG.2C, respectively. In some CS cells, calcium transients can be seenbefore observable contractions are noted, suggesting the development ofcardiomyocyte excitation elements in advance of maturing contractileelements.

Example 6 Distinguishing Spoc Cells from Bone Marrow Cells

[0155] Spoc cells are c-kit−, distinguishing them from the c-kit+ bonemarrow cells that have been used directly or indirectly in experimentsto reconstitute infarcted heart. Despite this, spoc cells could bederived from circulating bone marrow cells that become c-kit⁻ aftermigration to skeletal muscle. In order to more fully evaluate thisquestion, whole bone marrow was fractionated into c-kit+ and c-kit−populations. Both separate and combined populations were cultured underthe same conditions as spoc cells. None of the 3 marrow cell populationsdeveloped into spontaneously beating cells.

[0156] To test whether marrow cells have the potential to differentiateinto cardiomyocytes in the presence of soluble factors released fromspoc cells, equal proportions of marrow and spoc cells were co-culturedin a Costar transwell system, in which the two chambers are separated bya 0.4 μm permeable membrane. Although the total number of cellsincreased in each compartment, the spoc cells alone differentiated intobeating cells expressing cardiac markers.

[0157] In order to test if cell-cell contact between bone marrow andspoc cells would lead bone marrow cells to differentiate intocardiomyocytes, total bone marrow was mixed in equal proportion withEGFP-expressing spoc cells obtained from EGFP-expressing transgenic mice(ACTbEGFP, The Jackson Laboratory). In three separate experiments, underthe same culture conditions, total cell number increased, but onlyEGFP-expressing cells developed into beating cells. The converseexperiments showed a similar increase in cell number, but beating cellsdid not express EGFP. Taken together, these experiments show that bonemarrow does not contain any cell population phenotypically similar tospoc cells isolated from skeletal muscle.

Example 7 Distinguishing Spoc Cells from Cells Derived from the Heart

[0158] In order to determine if spoc cells can be isolated from heart,as well as skeletal muscle, the two tissues from the same mouse weredissociated and cultured separately. Only the spoc cell preparation fromskeletal muscle differentiated into beating cells expressing cardiacmarkers. Two replicate co-culture experiments of both cell populationsin Costar transwell systems produced an increased number of cells inboth chambers, but again, only the skeletal muscle-derived cellsdeveloped into beating cells expressing cardiac markers.

Example 8 Distinguishing Spoc Cells from Mesenchymal Stem Cells

[0159] To determine if spoc cells can be distinguished from mesenchymalstem cells (MSC), MSC were compared to spoc cells in culture. The MSC(Clonetics Corporation) were cultured in parallel with spoc cellsgenerated from skeletal muscle as described in the methods above. TheMSC adhered to the plate almost immediately upon plating, remainedadherent throughout 12 days of observation, and did not show any sign ofbeating. In contrast, the cardiac progenitor cells from skeletal musclewere smaller in size, remained nonadherent while they developed intofloating clusters of spoc cells, and they progressed to beating cardiacmyocytes expressing cardiac markers. Spoc cells did not form in the MSCcultures. Thus, spoc cells are not MSC.

Example 9 In Vivo Differentiation of Spoc Cells

[0160] In order to determine if spoc cells are capable in vivo ofcontinuing along the same differentiation pathway observed in vitro,approximately 100,000 EGFP-expressing, GATA-4-negative spoc cells wereinjected via tail vein into a mouse two months after an inducedmyocardial infarction (created by left coronary artery ligation). Twoweeks later, histologic examination of the heart showed EGFP positivecells that were now also GATA-4 positive, located in the peripheralregion of the infarct. Since spoc cells are not GATA-4 positive, thesefindings indicate that these cells can home to an area of cardiac damageand begin to differentiate into cardiomyocytes, as they do in vitro.

Example 10 Distinguishing Spoc Cells from Satellite Cells

[0161] Spoc cells and satellite cells are air-dried on glass slides for30 minutes and then fixed in 4% paraformaldehyde at 4° C. followed by arinse for 5 minutes with PBS. The cells are blocked with goat serum for30 minutes and then incubated overnight, at 4° C., with rabbit anti-met(1:200, Santa Cruz Biotechnology). Following the overnight incubation,the slides are rinsed 3 times (5 minutes each) with PBS and blockedagain with goat serum for 30 minutes. The cells are then incubated atroom temperature with a secondary antibody, conjugated with FluoresceinIsothiocyanate (FITC) for 1 hour. They are again rinsed 3 times (5minutes each) with PBS and then visualized with a laser confocalmicroscope (Leica) to detect fluorescent signals. Of the two cell typesexamined, only the satellite cells are positively stained with c-metindicating that satellite cells express c-met on their cell surface,whereas spoc cells do not.

Example 11 Method of Isolating Cardiomyocyte Precursor Cells from AdultHuman Skeletal Muscle

[0162] Skeletal muscle tissue is surgically obtained from the deltoidmuscle of an adult human, is cut into small pieces and is digested withcollagenase for two hours at 37° C. The digested tissue is cleared ofcell debris and other undigested tissue fragments by passage through a100 μm filter and then through a 40 μm filter. The cell suspension iscentrifuged at low speed to clear as much as of the small muscle fiberfragments as possible. The cells at this stage consist mostly ofclusters of small round cells approximately 4 μm in diameter which arethe human spoc cells. These cells do not express the satellite cellsurface marker c-met.

[0163] The spoc cells are plated at a density of approximately 10⁵ cellsper cm² in regular tissue culture dishes in complete growth medium (1:1DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 ng/ml humanEGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 μg/ml insulin, 5 μg/mltransferrin, 6 ng/ml selenium, 2 μg/ml ethanolamine (ITS-X, InvitrogenCorporation), 25 μg/ml gentamicin and 2.5 μg/ml fungizone (LifeTechnologies)). After a few days, the culture consists of a floatingpopulation of round cells and some adherent fibroblasts. The round cellsenlarge as they undergo a few rounds of cell division during which timethey become clusters of floating round cells with an increased diameterof 10-14 μm. The cells in these clusters were referred to as CS (cardiacprecursors from spoc) cells.

[0164] In view of the many possible embodiments to which the principlesof our invention may be applied, it should be recognized that theillustrated embodiments are only examples of the invention and shouldnot be taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. An isolated mammalian c-kit−/c-met− cardiomyocyte precursorcell of muscular origin.
 2. The cell of claim 1, wherein the cell is ahuman cell.
 3. The cell of claim 1, wherein the cell is a mouse cell. 4.The cell of claim 1, wherein the cell is from a fetus, a child, or anadult.
 5. The cell of claim 1, wherein the cell is in suspension.
 6. Thecell of claim 1, wherein the cell is between about 3 μm and 10 μm indiameter.
 7. The cell of claim 6, wherein the cell is approximately 4 μmin diameter.
 8. The cell of claim 1, wherein the cell differentiatesinto a cardiomyocyte.
 9. The cell of claim 1, wherein the celldifferentiates into a spontaneously beating cardiomyocyte.
 10. The cellof claim 1, wherein the cell is transduced with a viral vector.
 11. Thecell of claim 1 wherein the viral vector comprises a heterologousnucleic acid.
 12. The cardiomyocyte of claim 8, wherein thecardiomyocyte expresses GATA-4, troponin-T, L-type calcium channel, orNkx2.5, or a combination thereof.
 13. A method of isolating ac-kit−/c-met− cardiomyocyte precursor cell of muscular origin,comprising: separating cells of less than 40 μm in diameter from asuspension of muscle cells; culturing the cells in a tissue culturemedium on a solid substrate; and isolating the cells in suspension inthe medium; thereby isolating the c-kit−/c-met− cardiomyocyte precursorcell of muscular origin.
 14. The method of claim 13, wherein separatingcells of less than 40 μm in diameter from a suspension of cellscomprises: passing the suspension of cells through a first filter with apore size of about 50-200 μm to collect a first eluate containing cellsof greater than about 50 μm and less than about 200 μm in diameter; andpassing the first eluate through a second filter with a pore size ofabout 40 μm to collect a second eluate containing cells of less thanabout 40 μm in diameter.
 15. The method of claim 14 wherein the firstfilter has a pore size of at least 100 μm and the second filter has apore size of about 40 μm.
 16. The method of claim 13, wherein the tissueculture medium is a growth medium.
 17. The method of claim 16, whereinthe growth medium is supplemented with a growth factor.
 18. The methodof claim 17, wherein the growth factor is EGF, or bFGF, or a combinationthereof.
 19. The method of claim 18, wherein the growth factor EGF ispresent at a concentration between about 5 and 50 ng/ml.
 20. The methodof claim 19, wherein the growth factor EGF is present at a concentrationbetween about 5 and 10 ng/ml.
 21. The method of claim 19, wherein thegrowth factor EGF is present at a concentration of about 10 ng/ml. 22.The method of claim 18, wherein the growth factor bFGF is present at aconcentration between about 5 and 50 ng/ml.
 23. The method of claim 22,wherein the growth factor bFGF is present at a concentration betweenabout 5 and 10 ng/ml.
 24. The method of claim 22, wherein the growthfactor bFGF is present at a concentration of about 10 ng/ml.
 25. Amammalian c-kit−/c-met− cardiomyocyte precursor cell of muscular originisolated according to the method of claim
 13. 26. A method fordifferentiating a c-kit−/c-met− cardiomyocyte precursor cell of muscularorigin, comprising: separating cells of less than 40 μm in diameter froma suspension of muscle cells; culturing the cells in a tissue culturemedium in the presence of a growth factor on a solid substrate;isolating the cells in suspension in the medium; and removing the growthfactor, thereby differentiating the c-kit−/c-met− cardiomyocyteprecursor cell of muscular origin into a cardiomyocyte.
 27. The methodof claim 26, wherein the cardiomyocyte is spontaneously beating.
 28. Themethod of claim 26, wherein the growth factor is EGF, or bFGF, or acombination thereof.
 29. The method of claim 28, wherein the growthfactor EGF is present at a concentration between about 5 and 50 ng/ml.30. The method of claim 29, wherein the growth factor EGF is present ata concentration between about 5 and 10 ng/ml.
 31. The method of claim29, wherein the growth factor EGF is present at a concentration of about10 ng/ml.
 32. The method of claim 28, wherein the growth factor bFGF ispresent at a concentration between about 5 and 50 ng/ml.
 33. The methodof claim 32, wherein the growth factor bFGF is present at aconcentration between about 5 and 10 ng/ml.
 34. The method of claim 32,wherein the growth factor bFGF is present at a concentration of about 10ng/ml.
 35. A mammalian cardiomyocyte differentiated from a c-kit−/c-met−cardiomyocyte precursor cell of muscular origin according to the methodof claim
 26. 36. A method of treating a myocardial injury in a subject,comprising administering a therapeutically effective amount of the cellof claim 1, thereby treating the myocardial injury.
 37. The method ofclaim 36, wherein the cells are introduced locally into the myocardialinjury.
 38. The method of claim 36, wherein the cells are introducedsystemically into the subject.
 39. The method of claim 38, wherein thecells are introduced intravenously.
 40. The method of claim 36, whereinthe myocardial injury is cardiomyopathy, myocardial infarction orcongenital heart disease.
 41. A method of treating cardiac muscledysfunction, comprising administering to a subject with cardiacdysfunction a therapeutically effective amount of mammalianc-kit−/c-met− cardiomyocyte precursor cells of muscular origin thatdifferentiate into beating cardiomyocytes.
 42. The method of claim 41,wherein the cardiac muscle dysfunction is a myocardial infarction, acardiomyopathy, or a congenital heart disease.
 43. A pharmaceuticalcomposition comprising mammalian c-kit−/c-met− cardiomyocyte precursorcells of muscular origin in a pharmaceutically acceptable carrier.
 44. Amethod for screening for an agent to determine the effect of the agenton a cardiomyocyte comprising: providing mammalian c-kit−/c-met−cardiomyocyte precursor cells of muscular origin; contacting the cellswith the agent; and observing the effect of the agent on the cells. 45.The method of claim 44, wherein observing the effect comprisesdetermining the effect of the agent on differentiation of the cells. 46.The method of claim 45 wherein determination of the effect ondifferentiation comprises assaying expression of GATA-4, expression ofcardiac troponin-T, expression of L-type calcium channel, or expressionof Nkx2.5, or a combination thereof.
 47. The method of claim 45, whereinobserving the effect comprises assaying a parameter of cardiomyocytefunction of the cells.
 48. The method of claim 47 wherein the parametercomprises spontaneous beating of the cells.
 49. A kit for promotingcardiomyocyte differentiation, comprising a container containing apurified population of mammalian c-kit−/c-met− cardiomyocyte precursorcells of muscular origin.
 50. The kit of claim 49, further comprising acontainer containing a growth factor, a container containing a culturemedium, instructions for using the kit, or any combination thereof.